The neutrophil carries out its primary function of destroying foreign cells by a complex sequence that begins with binding of a stimulus to membrane receptors initiating membrane and cytoplasmic events that culminate in the activation of contractile elements, oxidative metabolism and degranulation. Microbicidal activity is achieved by the formation of reduced oxygen derivatives and secretion of granule enzymes. We will continue a detailed investigation of the process of activation of the metabolic burst in human neutrophils. The two major areas of study are the role of the Mol glycoproteins in initiation and regulation of the oxidative burst and characterization of the location, activation, and function of the superoxide-forming NADPH oxidase. We will compare O2 metabolism in normal cells, Mol-deficient cells, and normal cells treated with antibodies to Mol. The location, membrane orientation, and cytoskeletal association of Mol will be determined and its role in cytoskeleton- dependent events assessed. In studies of NADPH oxidase we will consider the involvement, subcellular location, and stimulus- indiced translocation of components such as cytochromeb559, flavoproteins, and quinones. Our recently described subcellular arachidonate-activatable oxidase system reconstituted from defined organelles of fractionated unstimulated cells will be characterized. Activation mechanisms will be defined considering roles for Mg2+, Ca2+, adenine and guanine nucleotides, N proteins, phospholipases, and protein kinases. A Mg2+-dependent cytosolic cofactor will be purified, characterized structurally, and used for antibody generation. Biosynthesis, processing and genetic regulation of this cofactor will be studied, cDNA probes developed, and the structural gene cloned. Membrane-associated oxidase components will be purified by detergent solubilization and substrate affinity chromatography. The proposed studies will elucidate cellular and molecular mechanisms of activation of phagocytes and provide valuable probes for further studies of stimulus-response coupling and clinical syndromes of neutrophil dysfunction.